Approximately 10% of Black American males experience hemolytic anemia after exposure to aniline and aniline-related drugs and environmental chemicals. Enhanced sensitivity is also seen in Americans of Mediterranean decent (Italian, Greek, Lebanese, etc.), where the hemolytic episode may be life-threatening. Extensive studies in the 1950's and 1960's revealed that sensitivity is associated with a relative deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G6-PD) and led to the concept that injury resulted from oxidative stress induced by metabolite(s) of aniline and related compounds. However, the hemolytic metabolite(s) were not identified and little is known as to their interaction with the RBC. Our long term objective is to determine the toxicological mechanisms underlying drug-induced hemolytic anemia. We have developed an animal model that shows a dose-dependent hemolytic anemia after aniline or dapsone. The hemolytic response appears to be similar to that of man. We have identified a metabolite of aniline (pnenylhydroxylamine), which is a) about 15 x more potent than aniline, and b) a direct acting hemotoxin, and hence may be solely responsible for the hemolytic anemia sen after aniline. The objectives of the present phase of the studies are a) to identify the hemolytic metabolites of aniline, phenacetin, dapsone, and primaquine; b) to identify the metabolic events which determine the formation and delivery of the ultimate toxins to the red cell and the relationship between delivery and response; and c) to examine the relevance of concepts developed in vitro with phenylhydrazine and other hematotoxins for the in vivo drug-induced hemotoxicity. The initial mechanism studies proposed here will begin to assess the metabolic basis for the dose/response curves, the significance of methemoglobin formation in the hemolytic sequence, and whether hemoglobin and/or the cell membrane can be eliminated as molecular targets for phenylhydroxylamine and related hemolytic drug metabolites. It is expected that these studies will provide the basis for a detailed toxicological assessment of the role of drug metabolism in drug-induced hemolytic anemia and further our understanding of mechanisms underlying this type of red cell injury in vivo.